Proteolysis plays an important role in numerous cellular processes. Alteration in the abundance of critical regulatory proteins frequently leads to a variety of diseases including several forms of cancer. In eukaryotes, conjugation of ubiquitin to many short-lived proteins is an obligatory step in their degradation. The Saccharomyces cerevisiae DOA4 gene encodes a deubiquitinating enzyme that is structurally related to the human tre-2 oncogene product. This enzyme is required for rapid degradation of ubiquitin-proteosome pathway substrates. In order to characterize Doa4 further, twelve extragenic suppressors of the doa4- 1 allele were isolated in a genetic screen aimed at the identification of spontaneous pseudorevertants. These involve seven different genes, of which six were cloned. Surprisingly, all six of these DID (Doa4-independent degradation) genes encode components of the vacuolar-protein-sorting (Vps) pathway. In particular, all of the proteins are class E Vps factors, which are necessary for maturation of a prevacuolar compartment into multivesicular bodies that then fuse with the vacuole. Genetic interaction between mutations in DOA4 and components of Vps pathway and physical interaction between corresponding proteins suggest that the enzyme is involved in protein sorting. Particularly, Doa4 may be responsible for deubiquitination of endocytosed membrane proteins prior to degradation in the vacuole. The main goal of this project is to characterize the relationship between the Vps pathway and the deubiquitinating enzyme Doa4. To achieve this aim, genetic and biochemical approaches will be used. Specifically: 1. Association of Doa4 with components of the Vps pathway will be studied by the methods of column chromatography and two-hybrid analysis. 2. Elements of the amino acid sequence of Doa4 involved in the interaction with components of the Vps pathway will be identified by site-directed mutagenesis. 3. Proteins necessary for suppression of the doa4 degradation defect by mutations of Vps factors will be identified by mutagenesis, using chemical and genetic approaches.